Serum Albumin
Serum albumin is the most abundant protein in mammalian sera (40 g/l; ≈0.7 mM in humans), and one of its functions is to bind molecules such as lipids and bilirubin (Peters T, Advances in Protein Chemistry 37:161, 1985). The half-life of serum albumin is directly proportional to the size of the animal, where for example human serum albumin (HSA) has a half-life of 19 days and rabbit serum albumin has a half-life of about 5 days (McCurdy T R et al, J Lab Clin Med 143:115, 2004). Human serum albumin is widely distributed throughout the body, in particular in the intestinal and blood compartments where it is mainly involved in the maintenance of osmolarity. Structurally, albumins are single-chain proteins comprising three homologous domains and totaling 584 or 585 amino acids (Dugaiczyk L et al, Proc Natl Acad Sci USA 79:71 (1982)). Albumins contain 17 disulfide bridges and a single reactive thiol, Cys34, but lack N-linked and O-linked carbohydrate moieties (Peters, 1985, supra; Nicholson J P et al, Br J Anaesth 85:599 (2000)). The lack of glycosylation simplifies recombinant expression of albumin. This property, together with the fact that the three-dimensional structure is known (He X M and Carter D C, Nature 358:209 (1992)), has made it an attractive candidate for use in recombinant fusion proteins. Such fusion proteins generally combine a therapeutic protein (which would be rapidly cleared from the body upon administration of the protein per se) and a plasma protein (which exhibits a natural slow clearance) in a single polypeptide chain (Sheffield W P, Curr Drug Targets Cardiovacs Haematol Disord 1:1 (2001)). Such fusion proteins may provide clinical benefits in requiring less frequent injection and higher levels of therapeutic protein in vivo.
Fusion or Association with HSA Results in Increased In Vivo Half-Life of Proteins
Serum albumin is devoid of any enzymatic or immunological function and, thus, should not exhibit undesired side effects upon coupling to a bioactive polypeptide. Furthermore, HSA is a natural carrier involved in the endogenous transport and delivery of numerous natural as well as therapeutic molecules (Sellers E M and Koch-Weser M D, Albumin Structure, Function and Uses, eds Rosenoer V M et al, (Pergamon, Oxford, p 159 (1977)). Several strategies have been reported to either covalently couple proteins directly to serum albumins or to a peptide or protein that will allow in vivo association to serum albumins. Examples of the latter approach have been described e.g. in EP 486 525 and U.S. Pat. No. 6,267,964, in WO01/45746 and in Dennis et al, J Biol Chem 277:35035-43 (2002). The first two documents describe inter alia the use of albumin-binding peptides or proteins derived from streptococcal protein G (SpG) for increasing the half-life of other proteins. The idea is to fuse the bacterially derived, albumin-binding peptide/protein to a therapeutically interesting peptide/protein, which has been shown to have a rapid clearance in blood. The thus generated fusion protein binds to serum albumin in vivo, and benefits from its longer half-life, which increases the net half-life of the fused therapeutically interesting peptide/protein. WO01/45746 and Dennis et al relate to the same concept, but here, the authors utilize relatively short peptides to bind serum albumin. The peptides were selected from a phage displayed peptide library. In Dennis et al, earlier work is mentioned in which the enhancement of an immunological response to a recombinant fusion of the albumin binding domain of streptococcal protein G to human complement receptor Type 1 was found. US Patent application no. 2004/0001827 (Dennis) also discloses the use of constructs comprising peptide ligands, again identified by phage display technology, which bind to serum albumin conjugated to bioactive compounds for tumour targeting. Whilst the constructs are said to have improved pharmacokinetic and pharmacodynamic properties, there is no disclosure or suggestion in this document of a reduction in the immunogenicity of the constructs compared to the unconjugated bioactive compounds. There is no suggestion that further serum albumin-binding conjugate molecules would be desirable.
As an alternative, the therapeutically interesting peptide/protein in question may also be fused directly to serum albumin, as mentioned above and described by Yeh et al (Proc Natl Acad Sci USA 89:1904 (1992)) and Sung et al (J Interferon Cytokine Res 23:25 (2003)). Yeh et al describe the conjugation of two extracellular Ig-like domains (V1 and V2) of CD4 to HSA. The HSA-CD4 conjugate has a retained biological activity of CD4, but the half-life was reported to increase 140 times in an experimental rabbit model, as compared to CD4 alone. Soluble CD4 alone has an elimination half-life of 0.25±0.1 hrs, whereas the elimination half-life of HSA-CD4 was reported to be 34±4 hrs. A prolonged elimination half-life was also observed for interferon-beta (IFN-β) upon conjugation to HSA, as outlined by Sung et al, supra. Here, IFN-β-HSA conjugate was evaluated in primates, and the half-life of IFN-β was reported to increase from 8 hrs alone to 36 to 40 hrs when conjugated to HSA.
Albumin Binding Domains of Streptococcal Protein G
Streptococcal protein G (SpG) is a bifunctional receptor present on the surface of certain strains of streptococci and is capable of binding to both IgG and serum albumin (Björck L et al, Mol Immunol 24:1113 (1987)). The structure is highly repetitive with several structurally and functionally different domains (Guss B et al, EMBO J. 5:1567 (1986)). More precisely, SpG comprises one Ig-binding motif and three serum albumin binding motifs (Olsson A et al, Eur J Biochem 168:319 (1987)).
The albumin-binding protein BB, derived from streptococcal protein G, has 214 amino acid residues and contains about 2.5 of the albumin-binding motifs of SpG (Nygren P-Å et al, J Mol Recognit 1:69 (1988)). It has been shown previously that BB has several properties that make it highly suitable as a fusion partner for peptide immunogens with the purpose of creating powerful vaccines. For example, BB has been fused with repeated structures from the P. falciparum malaria antigen Pf155/RESA (M3) (Sjölander A et al, J Immunol Meth 201:115 (1997)), and to a respiratory syncital virus (RSV) (Long) G protein fragment (G2Na) (Power U F et al, Virol 230:155 (1997)). Both BB-M3 and BB-G2Na were able to trigger strong and long-lasting antibody responses against both immunogen fusion moieties in several animal models. BB-M3 induced high titres of antibodies in rabbits after covalent conjugation to immune stimulating complexes (iscoms) (Sjölander A et al, Immunometh 2:79 (1993)) and is immunogenic in mice (Sjölander et al, 1997, supra) and Aotus monkeys (Berzins K et al, Vaccine Res 4:121 (1995)). The observed effect was observed in the presence of a potent adjuvant, e.g. Freund's complete adjuvant (FCA). Furthermore, BB-G2Na induced detectable and protective antibody responses in both mice and man (Power et al, 1997, supra; Power U F et al, J Infect Dis 184:1456 (2001)). In agreement with BB-M3, this effect was observed in the presence of a strong adjuvant, in this case mannitol and aluminum phosphate.
The structure of one of the serum albumin-binding motifs of SpG, designated A3, ABD3 or just ABD (“albumin binding domain”), has been determined (Kraulis P J et al, FEBS Lett 378:190 (1996)). This study revealed a three-helix bundle domain, surprisingly similar in structure to the Ig binding domains of staphylococcal protein A. The SpG domain ABD corresponds to 46 amino acids.
The albumin binding parts of SpG have been epitope mapped closely, as described by Goetsch et al (Clin Diagn Lab Immunol 10:125 (2003)).
Other Albumin-Binding Domains
Albumin-binding proteins are found in other bacteria. For example, naturally occurring albumin-binding proteins include certain surface proteins from Gram+ bacteria, such as streptococcal M proteins (e.g. M1/Emm1, M3/Emm3, M12/Emm12, EmmL55/Emm55, Emm49/EmmL49 and Protein H), streptococcal proteins G, MAG and ZAG, and PPL and PAB from certain strains of Finegoldia magna (formerly Peptostreptococcus magnus). See review of Gram+ surface proteins by Navarre W W and Schneewind O (Microbiol Mol Biol Rev 63:174-229 (1999)), and references contained therein. The characteristics of albumin-binding by some of these proteins have been elucidated further, by e.g. Johansson M U et al (J Biol Chem 277:8114-8120 (2002)); Linhult et al (Prot Sci 11:206-213 (2002), and Lejon S. et al J. Biol. Chem. 279, 41, 2004, 42924-42928).
Clinical Implications of Immunogenicity
Most biologically active proteins, including proteins that are more or less identical to proteins native to the species in question, induce antibody responses upon administration to a significant fraction of subjects. The main factors that contribute to immunogenicity are presence of foreign epitopes, e.g. new idiotopes, different Ig allotypes or non-self sequences, impurities and presence of protein aggregates. In the majority of cases, the induced antibodies have no biological or clinical effects. Where a clinical effect is observed, the most common is a loss of efficacy of the biopharmaceutical.
However, cases with more serious adverse events have been reported. On these occasions, antibodies raised against a protein pharmaceutical cross-reacted with endogenous proteins. Erythropoietin is such an example. When administering erythropoietin to humans, immune responses were induced that led to pure red cell aplasia in the patients (Casadevall N et al, New Eng J Med 346:469 (2002)). The specific antibodies that were generated were of high affinity, and were also shown to cross-react with other forms of erythropoietin such as Eprex®, Epogen® and NeoRecormon®, which indicates that the reactivity was most likely directed against the erythropoietin active site conformation. Another example is thrombopoietin, which, upon administration to humans, resulted in the production of neutralizing antibodies. The antibodies inhibited the activity of endogenous thrombopoietin, which resulted in autoimmune thrombocytopenia (Koren E et al, Curr Pharm Biotech 3:349 (2002)).
Given that the clinical use of biopharmaceuticals often elicits an immune response, immunogenicity is a risk factor to be managed during the development of all biopharmaceutical products. Besides erythropoietin and thrombopoietin mentioned above, several other biopharmaceuticals have also been reported to induce immune responses in patients. Examples are ciliary neurotrophic factor (CNTF), granulocyte-macrophage colony-stimulating factor (GM-CSF), growth hormone (GH), insulin and interferon-beta (IFN-β). The reasons why the above proteins are observed to generate antibodies in the treated patients differ depending on the product. More precisely, the main factor in the immunogenicity of insulin appeared to have been protein impurities which acted as adjuvants, whereas in the case of IFN-β the main factors were believed to be lack of glycosylation when the protein was produced in a bacterial host cell and presence of aggregates due to low solubility (Karpusas M et al, Cell Mol Life Sci 54:1203 (1998)). Before the advent of recombinant human GH, GH from human cadavers was used to treat GH-deficiency in children. Mainly due to a high content of protein impurities, 45% of the children produced antibodies against this first generation of products (Raben M S, Recent Prog Horm Res 15:71 (1959)). When recombinant GH, which includes an extra methionine residue that enables production in E. coli, was administered to the patients, the incident of antibodies decreased to 8.5% (Okada Y et al, Endocrinol Jpn 34:621 (1987)). The immunogenicity of GH is more complex than percent identity to the self-protein or lack of immune tolerance, judging from the fact that only one twin developed antibodies to recombinant GH when twins with homozygous GH deletion mutants were treated with the therapeutic protein (Hauffa B P et al, Acta Endocrinol 121:609 (1989)). Recombinant human CNTF was produced with the purpose of treating patients with amyotrophic lateral sclerosis (ALS), since CNTF is believed to enhance the survival of motor neurons. Unfortunately, more than 90% of these patients were tested positive for anti-CNTF antibodies after two weeks of treatment. The clinical effect of the therapeutic protein was severely hampered by the specific antibodies, as shown by the ALS CNTF Study Group report in 1995 (Clin Neuropharmacol 18:515 (1995)). A second generation of CNTF, which contains a truncated C-terminus and is PEGylated, is currently under development.
Furthermore, the immunogenicity of therapeutic antibody molecules is a significant problem which severely limits their widespread and repeated application in treating many diseases.
Different Strategies to Decrease Immunogenicity
Technologies that reduce immunogenicity of proteins are therefore needed. Actually, the importance of such technologies is increasing, since it is becoming more and more common that protein pharmaceuticals have amino acid sequence modifications compared to the naturally occurring protein, or are altogether comprised of amino acid sequences foreign to the subject. One important method to prevent immunogenicity is by optimization of production, purification and formulation of the biopharmaceutical protein to generate soluble, non-aggregated, native protein which is free of contaminating adjuvants. There are several reports on the reduction of immunogenicity of proteins, e.g. human growth hormone (Moore W V et al, J Clin Endocrin Meth 51:691 (1980)) and interferon-α 2a (Hochuli E, J Inter Cyto Res 17:15 (1997)), through improvement of purification and formulation.
Other methods to alter immunogenicity are directed against the actual sequence or structure of the protein in question, and sometimes referred to as “deimmunization methods”. Examples of such strategies are epitope neutralization, gene-shuffling, chemical modifications and immune tolerance. Epitope neutralization involves rational identification of dominant T and/or B-cell epitopes using in silico and/or in vitro methods, and subsequent redesign of highlighted sequences to eliminate the dominant epitopes and, hopefully, obtain decreased immunogenicity (Stickler M et al, J Immunother 6:654 (2000); US Patent Application Publication No. 2003/0166877). Another example of deimmunization and gene-shuffling is the humanization of antibody molecules (Kuus-Reichel K et al, Clin Diagn Lab Immunol 1:365 (1994)). The immunogenicity has been reported to drop going from murine to chimeric to fully human antibodies. The evolution of human proteins using DNA gene-shuffling involves homology-dependent recombination of DNA fragments to generate ordered chimaeras of genes. Gene-shuffling could be useful when seeking proteins with reduced immunogenicity and with retained biological activity (Pavlinkova G et al, Int. J. Cancer 94:717 (2001)).
Another method to alter the antigenicity (binding to pre-existing antibodies) and immunogenicity (ability to induce new immune responses) of a protein is to modify the protein chemically. Chemical modifications can be accomplished using covalently bound polymers such as polyethylene glycol (PEG) (Molineux G, Pharmacother 23:3 (2003)) and Dextran (Kobayashi K et al, J Agric Food Chem 49:823 (2001)), or performing neutralization of positive charges with succinic anhydride. PEG is a non-toxic, highly soluble molecule that has been shown to increase the half-life in vivo of proteins covalently bound thereto, and to reduce the immunogenicity of such proteins (Molineux G, supra). The PEG approach is commonly referred to as “PEGylation” of a protein.
Induction of immune tolerance offers a more acceptable means for preventing an immune response than PEGylation, since no chemical additions are made to the therapeutic molecule. The same pharmaceutical is administered, which e.g. ensures a better patient compliance. This approach has been tried for example in the context of administration of factor VIII to patients suffering from hemophilia A. One complication in using factor VIII to treat hemophilia A is the generation of inhibitory antibodies to the therapeutic protein, which is observed in about one-third of all patients (Scharrer I, Haemophilia 5:253 (1999)). Daily injections of large doses of factor VIII together with immunosuppressive agents, which should be given for time periods of from months to years is one strategy that is being pursued in the effort of trying to limit the immune response.
Drawbacks of Current Strategies for Reducing Immunogenicity
The drawbacks of the different approaches to reduce immunogenicity are several. For one thing, it is difficult to achieve covalent attachment to a protein of PEG molecules without obstructing the active sites that are essential for drug efficacy. Avoiding this is a major challenge in PEGylation. There is a great variation in the quality of PEGylated products, and numerous factors have shown to play a part in this variation: the presence or absence of linkers between PEG and the protein; the nature and stability of the bond(s) between the PEG, linker and protein; the impact of PEG attachment on surface charge of the resulting PEGylated protein; the coupling conditions; the requirement of proving that the product is homogeneous; and the relative toxicity of the activated polymer. Moreover, considerable modifications of the prototype method, and also a process of biological optimization have been required to achieve good results in terms of conservation of bioactivity. Any reduction in activity has to be addressed by increasing the treatment dosage, which once again increases the risk of an immune reaction to those molecules. Another drawback of the PEGylation approach is that PEGylated therapeutics increase the cost of treatment by an estimated USD 1000 per month per patient. There has been very little success with polymers other than PEG with regard to improving the pharmacological and immunological properties of therapeutic protein molecules (Burnham N L, Am J Hosp Pharm 51:210 (1994)).
As stated above, the immunogenicity of therapeutic antibody molecules has been addressed using the humanization approach. Humanization has worked well for some murine antibodies, e.g. HERCEPTIN®, which is approved for treatment of some breast cancers. In other cases, humanized antibodies, e.g. CAMPATH®-1H used for treatment of rheumatoid arthritis, still induce an immune response in 60% of the treated patients. Additionally, data from animal studies have shown that rodents are clearly not tolerant of antibodies from the same species and strain (Cobbold S P et al, Meth Enzym 127:19 (1990)) and fully human antibodies are believed to have the potential to evoke anti-idiotypic antibodies just like any other antibody.
Furthermore, the “deimmunization” approach, for example the targeted elimination of T and B-cell epitopes, is not as trivial as it may seem. The algorithms that are available for in silico prediction of epitopes may not be reliable. In the case of predicting B-cell epitopes, this is fairly difficult to do using algorithms, since such epitopes to a great extent are conformational epitopes. T-cell epitopes, on the other hand, are linear, which means that the existing in silico tools are more reliable. Unfortunately, most algorithms are suitable for identifying major histocompatibility complex (MHC) class I associated peptides and not MHC class II associated peptides. Since the latter are more relevant for T-helper cell activation, this is a drawback when seeking to reduce antibody responses. Furthermore, the great polymorphism of MHC molecules makes it difficult to predict, using immunoinformatics, the majority of the T-cell epitopes of any given protein antigen. It is important to remember that epitopes identified by immunoinformatics should always be verified by experimental studies in e.g. in vitro human T-cell stimulation assays. One of the reasons for this is that binding of an immunogenic peptide (i.e. a T-cell epitope) to MHC class II molecules is not sufficient to ensure recognition by a given T cell antigen receptor that has specificity for the peptide. The studies that are necessary for the identification of both T and B-cell epitopes are time-consuming as well as experimentally difficult.
The major disadvantages involved in inducing tolerance to different therapeutics, such as factor VIII exemplified above, are the effects of long-term treatment with immunosuppressive agents (such as sensitivity to infections following suppression of the immune system, and potential toxic effects of the agents) and the high cost involved. It has been estimated that the induction of immune tolerance against factor VIII in a paediatric patient costs nearly USD 1 million.
Despite the existence of strategies for reducing the immunogenicity of biopharmaceuticals and other proteins with biological activity, none of these strategies has proved itself useful in all situations where the reduction or elimination of immunogenicity is desired. Thus, there is a continued need for complementary approaches to the problem.